HIV-1 is the causative agent of AIDS. The three viral enzymes - RT, IN, and protease (PR) - have essential roles in the replication of HIV-1 and are the targets for all of the most potent anti-HIV drugs. Although considerable progress has been made in treating HIV-infected patients with three- and four-drug regimens, there is an immediate need for the development of effective ways to prevent new infections. A potent preventive vaccine would be ideal; however, despite a huge effort, the goal of developing an effective vaccine remains elusive. In the absence of an effective vaccine, reducing the transmission of HIV-1 must rely on barrier methods and/or drug treatments. There are two ways that anti-HIV drugs can be used to reduce viral transmission: (1) effective therapy in infected patients can reduce the viral load, making it less likely that an infected individual will transmit the virus to a partner; and (2) treating the uninfected partner with an anti-HIV drug can block transmission. Because most new infections are caused by a single virus, blocking transmission is an attractive option and there is now good evidence that giving an anti-HIV drug to the uninfected partner can significantly reduce viral transmission if the uninfected partner is compliant. Because of the problem of drug resistance, it would be better to use drugs with nonoverlapping resistance profiles for treatment and prophylaxis. Treatment would have to be long term and, for this reason, drug toxicity is an important consideration, which argues against the use of nucleoside RT inhibitors (NRTIs). It would also be better to block infection before the viral DNA is integrated, which argues against the use of PR inhibitors. Therefore, the two remaining options among the major classes of anti-HIV drugs are nonnucleoside RT inhibitors (NNRTIs) and IN strand-transfer inhibitors (INSTIs). We are using a combined approach that involves structural analysis, biochemistry, virology, modeling, toxicity testing, and chemistry to design, synthesize, and evaluate new NNRTIs and INSTIs. We have made good progress in developing new compounds that are effective against the wild-type (WT) and common drug-resistant viruses and that have good therapeutic indexes in tests done in cultured cells. Our progress with the IN research is reported below; progress with the HIV-1 RT research is reported separately for Project ZIA BC 010481. HIV-1 IN. Like RT, HIV-1 IN is an important drug target; however, as is the case for all anti-HIV drugs, treatment with INSTIs leads to resistance. We are making good progress on two fronts: understanding how mutations in IN confer resistance to the currently available compounds, and developing new INSTIs that are effective against the common drug-resistance mutations. Dr. Terry Burke is synthesizing new anti-IN compounds; Dr. Yves Pommier is using biochemical assays to test Dr. Burke's anti-IN compounds in vitro (using purified recombinant IN); and we are testing how the new inhibitors affect viral replication and measuring their toxicity in cultured cells. Until quite recently, we had no structural information to guide the development of IN inhibitors. However, Dr. Peter Cherepanov has obtained high-resolution structures of full-length foamy virus (FV) IN in complexes with both DNA substrates and anti-IN drugs. Dr. Cherepanov has joined our collaborative effort and has solved the structures of FV IN in complex with some of the more promising compounds developed by Dr. Burke. The active site of FV IN is similar, but not identical, to the active site of HIV-1 IN, and we are using Dr. Cherepanov's data to develop models of HIV-1 IN (both WT and mutant). These models have helped us understand how resistance arises and have been useful in the design of more effective compounds. Dr. Burke has recently synthesized several novel compounds that have IC50s in the low nanomolar range in a one-round replication assay, and that effectively inhibit both WT IN and most of the common drug-resistant variants. Based on tests done in cultured cells, these compounds have excellent therapeutic indexes (the CC50s are more than 3 logs higher than the IC50s). We also have three projects that involve studies of HIV-1 integration. In the first project, we are working with Drs. Alan Engelman and Vineet KewalRamani to define the host factors that are involved in transporting the preintegration complex (PIC) and to determine the exact roles they play in this process. In the second project, we are taking advantage of the fact that it is possible to redirect where HIV-1 DNA preferentially integrates. Redirecting HIV-1 DNA integration has the potential to make gene therapy safer because it may help solve the problems associated with the insertional activation of oncogenes; in addition, the technology can be used to determine where in the genome proteins/domains bind to chromatin. Different retroviruses have different integration-site preferences. There are good reasons to believe that such preferences are based on which host factor(s) the PIC interacts with; however, in most cases, it is unclear what the host factor(s) might be. Lentiviruses (including HIV-1) are the exception: HIV-1 IN is known to bind to lens epithelium-derived growth factor (LEDGF); the distribution of LEDGF on chromatin is the major factor that determines the local sites where HIV-1 DNA integrates. However, working with Dr. KewalRamani, we recently found that the host factor CPSF6, which binds to the viral CA protein, helps direct the PIC to nuclear speckles, which are associated with regions of the genome that are enriched in highly expressed genes. Thus, the interaction of CA and CPSF6 directs the PIC to broad regions of the genome, and the interaction of LEDGF and IN determines the exact sites where the HIV DNA is integrated. We, and others, showed that replacing the N-terminus of LEDGF with chromatin-binding domains (CBDs) from other proteins changes the specificity of HIV-1 DNA integration. The initial experiments were done either with single CBDs or, in one case, with two linked domains taken from a larger protein. These analyses showed that the binding sites for CBD can be accurately determined by mapping redirected HIV-1 integration sites and that the distance between the CBD binding site and the integration site(s) is relatively small. We also showed that the binding sites for multiple-domain modules reflect the combinatorial interactions of the individual domains and chromatin and that the structural relationship of the domains helps define binding specificity. We have recently moved from an analysis of isolated CBDs to intact proteins. Although not every protein we have tried has worked, we have been able to get excellent data with a number of relatively large proteins, including TAF3, and we have been able explore how TAF3 interacts with its binding sites on chromatin using mutants with altered binding specificities (the TAF3 experiments are part of a collaboration with Dr. Robert Roeder). More recently, we have mapped the genome-wide distribution of the binding sites for the proteins in the integrator complex (with Drs. J. Skaar and M. Pagano) and WT and mutant DNMTs (with Drs. M. Noh and D. Allis). In the third project, we are working with Drs. Xiaolin Wu, Frank Maldarelli, John Coffin, John Mellors, and Mary Kearney to determine the distribution of integration sites in HIV-infected patients. We recently showed that there is extensive clonal expansion of HIV-infected cells in patients, and that, in some cases, integration of HIV DNA in specific oncogenes (MKL2 and BACH2) can contribute to this clonal expansion.